The synthesis method of oligonucleotide includes a phosphate triester method, an H-phosphonate method, a phosphoramidite method and the like, and solid phase synthesis (solid phase method) using a phosphoramidite method is most widely used at present (non-patent document 1). The solid phase method is advantageous from the aspect of speed, since process has been optimized and automation has progressed. However, it is associated with defects in that scaling-up is limited due to facility restriction, reagents and starting materials are used in excess, and confirmation of the progress status of the reaction in an intermediate step, analysis of intermediate structure and the like are difficult.
On the other hand, synthesis methods of oligonucleotide by a liquid phase method have also been studied. However, since the operation is complicated and the yield is low, a large-scale, rapid synthesis of long oligonucleotide is difficult.
In recent years, in an attempt to solve the respective defects of the liquid phase method and the solid phase method, an oligonucleotide production method using monomethoxypolyethylene glycol (MPEG) as a protecting group is disclosed (non-patent documents 2 to 4). However, while synthetic examples of up to 20 mer DNA are disclosed, a crystallization isolation operation is essential for each reaction, and the progress status of the reaction and the like are difficult to confirm, since MPEG molecule itself is not a unimolecule.
In the meantime, a synthesis method of oligonucleotide comprising use of a hydrophobic group-linked nucleoside is disclosed (patent document 1). While it has been reported that the method affords synthesis of 21 mer oligonucleotide, the number of steps is markedly high and they are complicated, since a crystallization isolation operation is repeated in every step of deprotection of 5′-protecting group, coupling and oxidation.